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ADAM15/PTK6/Cmet Interplay: Promotors of Prostate Cancer

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ADAM15/PTK6/Cmet Interplay: Promotors of Prostate Cancer ADAM15/PTK6/cMET interplay: Promotors of prostate cancer progression Melanie Hurtz, MSc. September 2017 School of Medicine Cardiff University Cardiff, CF14 4XN Cardiff, United Kingdom A thesis submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy (PhD) DECLARATION This Work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other aWard. Signed ………………………………… (candidate) Date ………………………… I STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD Signed ………………………………………(candidate) Date ………………………… STATEMENT 2 This thesis is the result of my oWn independent Work/investigation, except Where otherWise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………(candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………(candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ………………………………………(candidate) Date ……………………… II Σε αυτό που βρήκα εδώ στο καρντιφ, και το μικρό μπόνους Für Joko, und all die guten Tage zum Fliegen Für Mama und Papa III Acknowledgments This thesis Would have been impossible Without the support of my supervisor’s Dr Zara Poghosyan and Dr Vera Knäuper. …. and also Without the support and great organization from the people behind the scene, Julia, Trish, Marie and AJ. I Would like to say thank you to Dr Lisa Spary from the Wales Cancer Bank, for the great sample coordination. I Would like to say thank you to my sponsor, Cancer Research Wales, for supporting this project. A special thank you also to all unknoWn patients, that Were Willing to support prostate cancer research, only With you We can try to find a Way to beat cancer. IV Abstract ADAM15 is a transmembrane metalloproteinase involved in disease progression and aggressiveness in prostate cancer (PCa). ADAM15 is composed of an extracellular domain, a transmembrane region and an intracellular domain, the latter being subject to splicing due to alternative use of exons 19 to 21. The splice variants that Will be subject in this study are ADAM15-A, ADAM15-B, ADAM15-C, ADAM15-D and ADAM15-E. Previously, ADAM15 splice variant A and B Were reported to associate With the PCa promotor PTK6 and the cMET adaptor protein Grb2. In order to understand the underlying mechanisms for the contribution of ADAM15 to disease progression in PCa, ADAM15 A-E splice variants, overexpressed in LNCaP and PC3 PCa cells, were biochemically and functionally characterized. Overexpression of ADAM15-A in PC3 led to enhanced invasion upon HGF treatment, which could be reverted by cMET inhibitor treatment. In addition, ADAM15-induced invasion Was dependent on its proteolytic activity. Moreover, PC3 cells expressing proteolytically active ADAM15 shoWed more MMP2 activity compared to cells With the proteolytically inactive ADAM15 mutant in cell supernatants. In contrast to the aggressive, androgen independent PC3, androgen dependent LNCaP cells did not shoW any response to HGF treatment upon ADAM15 A-E overexpression. All ADAM15 splice variants Were found in a complex With PTK6, which could be disrupted upon cMET inhibition in PC3. Strikingly, ADAM15 Was found in a complex With cMET/Gab1/Grb2/PTK6. cMET inhibition led to complex loss of cMET/Gab1/PTK6, hoWever, Grb2 remained in complex with ADAM15 regardless of treatment. Unlike cMET, PTK6 activity was not needed for formation of the ADAM15 complex. Analysis of the ADAM15 splice profile in prostate cancer patients and comparison With healthy prostate tissue revealed a significant overexpression of all ADAM15 splice variants. In summary, We shoW for the first time that ADAM15 is found in a complex With cMET/Gab1/Grb2/PTK6, and importantly, that, this complex formation is dependent on the cMET/HGF axis in PC3 PCa cells. Moreover, We found that proteolytically active ADAM15 resulted in enhanced invasion upon HGF treatment in PC3s. Our data suggest an important role for ADAM15 in prostate cancer disease progression. V Table of contents Acknowledgments .................................................................................................. IV Abstract .................................................................................................................... V Overview of Figures and Tables ........................................................................... IX List of abbreviations ............................................................................................ XIII 1 Introduction ...................................................................................................... 1 1.1 Cancer ........................................................................................................ 1 1.2 Prostate Cancer ........................................................................................ 2 1.2.1 The Prostate ........................................................................................ 3 1.2.2 PCa screening ..................................................................................... 3 1.2.3 Prostate cancer grading, the Gleason Score ....................................... 4 1.2.4 Androgen dependent PCa development .............................................. 5 1.2.5 Androgen independent PCa ................................................................ 6 1.2.6 Alternative splicing, a promotor of prostate cancer? ............................ 9 1.3 Zinc Metalloproteinases ......................................................................... 10 1.3.1 Matrix metalloproteinases .................................................................. 11 1.3.2 ADAMs ............................................................................................... 11 1.3.3 ADAM9 ............................................................................................... 16 1.3.4 ADAM10 ............................................................................................. 16 1.3.5 ADAM17 ............................................................................................. 18 1.4 ADAM15 ................................................................................................... 19 1.4.1 ADAM15 discovery ............................................................................ 19 1.4.2 ADAM15 gene structure .................................................................... 19 1.4.3 The modular structure of ADAM15 .................................................... 20 1.4.4 ADAM15 alternative exon use of the ICD .......................................... 24 1.4.5 Identified interaction partners of the ADAM15 ICD ............................ 25 1.4.6 Substrates for ADAM15 ..................................................................... 27 1.4.7 ADAM15 splice variants as biomarkers ............................................. 29 1.5 Protein Tyrosine kinases ....................................................................... 30 1.6 RTKs ......................................................................................................... 30 1.6.1 RTK modular organization ................................................................. 30 1.6.2 RTK activation.................................................................................... 33 1.6.3 The HGFR family ............................................................................... 33 1.6.4 The cMET/HGF axis .......................................................................... 39 1.7 Intracellular PTKs ................................................................................... 44 1.7.1 SRC ................................................................................................... 45 1.7.2 Scr-like tyrosine kinases, the FRK family .......................................... 46 1.7.3 PTK6 .................................................................................................. 46 1.7.4 PTK6 role in prostate cancer ............................................................. 50 1.8 Work leading to this project ................................................................... 51 1.9 Aims of the project .................................................................................. 51 2 Materials and Methods ................................................................................... 53 2.1 Cell Culture .............................................................................................. 53 2.1.1 Subculturing of cell lines .................................................................... 53 2.1.2 Freezing and thaWing of cell lines ...................................................... 54 VI 2.1.3 Stable transfection of PC3 and LNCaPs using lentiviral transduction 54 2.1.4 ADAM15 proteolytic activity E349A mutant ....................................... 55 2.1.5 MDA-MB-231 breast cancer cells ...................................................... 55 2.1.6 Stable transfection of PC3 With shRNA encoding plasmids .............. 55 2.1.7 Transient
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